CN114288278B

CN114288278B - Medicine-carrying microalgae, preparation method and application thereof

Info

Publication number: CN114288278B
Application number: CN202111375957.0A
Authority: CN
Inventors: 周民; 钟丹妮; 张东晓
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2023-12-05
Anticipated expiration: 2041-11-19
Also published as: CN114288278A

Abstract

The invention discloses a medicine-carrying microalgae, a preparation method and application thereof. The medicine-carrying microalgae is prepared by taking spirulina platensis as a medicine carrier and directly carrying the medicine curcumin through a one-step method. The method is simple, feasible, green, safe, large in drug loading rate and high in drug loading efficiency. The drug-loaded microalgae is gradually degraded at intestinal tract parts after being orally taken, and slowly releases drugs, so that the concentration of the drugs in intestinal tracts is obviously improved. The oral administration of the drug-loaded microalgae preparation can combine radiotherapy and chemotherapy, synergistically inhibit tumors, and effectively prevent radiation damage of intestinal tissues; in addition, oral administration of the drug-loaded microalgae can effectively reduce inflammatory reaction of ulcerative colitis; the drug-loaded microalgae has excellent performance in fluorescence imaging, and can realize noninvasive tracking and real-time monitoring of drugs in vivo; the drug-loaded microalgae can be degraded through gastrointestinal tracts, and shows good biodegradability; the invention has wide application prospect in realizing oral drug delivery and imaging guidance treatment of intestinal related diseases by using bioactive materials.

Description

Medicine-carrying microalgae, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a curcumin-loaded spirulina platensis, a preparation method and application thereof.

Background

Oral administration remains the preferred and most common route of administration for the treatment of gastrointestinal disorders, mainly due to the high safety and patient compliance of the oral administration regimen, and the ease of manufacture, storage and transportation of the oral drug. However, oral drugs still face many challenges, including the susceptibility of oral drugs to degradation in gastric acid environments, and poor retention and bioavailability of many drugs in the intestinal tract. In order to increase the efficiency of oral administration, various drug carriers are currently being studied to design novel oral administration systems such as liposomes, dendrimers, micelles, polymer conjugates, polymer nanoparticles, silicon or carbon materials, and metal and magnetic nanoparticles. In general, complex design ideas and complicated synthesis methods are required to adjust chemical and physical characteristics of the above-mentioned drug carriers, thereby realizing versatility of the drug delivery system and being applied to drug delivery and disease treatment diagnosis. This also causes major problems such as technical challenges, high manufacturing costs, and low manufacturing efficiency. More importantly, the feasibility of converting these chemically synthesized materials into clinical applications is severely limited by inadequate biodegradability, low stability and in vivo non-specific toxicity. Therefore, how to develop a reasonable, effective and biocompatible multifunctional oral delivery way or system to improve the curative effect of oral drugs and exert the diagnosis and treatment effects of gastrointestinal diseases is a key problem to be solved urgently.

Based on this, drug delivery systems constructed with natural biological materials hold great promise for oral treatment of gastrointestinal disorders. Microalgae, which is a natural biological material, has abundant biological resources in nature, and has shown important application value in the fields of food, medicine, aquaculture, energy, agriculture, environmental protection and the like. Among them, spirulina platensis used in the present invention has extremely high nutritional components such as phycocyanin, carotenoid and polysaccharide, etc., and has been developed into an oral nutritional dietary supplement. Moreover, spirulina platensis is biodegradable in a physiological environment, providing a degree of biosafety for its application in biomedical fields. It has been shown that negatively charged surfaces of spirulina platensis can be loaded with positively charged small molecule drugs by electrostatic adsorption, and that continuous water channels and connecting pores (14-16 nm) in their cell membranes also allow small molecules to pass through and into the membrane. The search of the design of oral administration system and the treatment of gastrointestinal tract diseases at home and abroad and the patent result show that: there have been no reports of oral preparations based on curcumin-loaded spirulina platensis and their use.

Disclosure of Invention

The invention aims to provide a natural, environment-friendly, simple and feasible and easily-scaled drug-loaded microalgae, a preparation method and application thereof in treating intestinal diseases and the like.

The technical scheme adopted by the invention is as follows:

a drug-loaded microalgae consisting of spirulina platensis and curcumin loaded in the spirulina platensis.

Further, the curcumin loading rate is greater than 80%.

The preparation method of the drug-loaded microalgae specifically comprises the following steps:

adding curcumin solution into ultrapure water suspended spirulina platensis algae liquid, stirring and incubating in dark, loading curcumin into spirulina platensis by using the active surface of microalgae, and separating precipitate to obtain the drug-loaded microalgae.

Further, the proportion range of the ultrapure water suspended spirulina platensis algae liquid and the curcumin solution is 50-500 mug/mL, 20-2000 mug/mL.

The drug-loaded microalgae has high drug-loading efficiency, high drug-loading capacity and high biosafety, greatly improves the utilization efficiency and biocompatibility of a drug-loading system in organisms, and can be used as a functional oral drug delivery system comprising curcumin such as blood fat reduction, tumor resistance, inflammation resistance, cholagogue, oxidation resistance and the like for treating diseases; wherein, the inherent chlorophyll in the spirulina carrier has natural fluorescence characteristics, can realize gastrointestinal tract fluorescence imaging after oral administration, can realize noninvasive tracking in vivo without any extra fluorescent label, namely: can be used as contrast agent for fluorescence imaging of gastrointestinal tract.

Furthermore, the drug-loaded microalgae can be used for carrying out different doses according to early imaging diagnosis of tumor patients and enteritis patients. The dosage and concentration of the curcumin spirulina platensis as a medical imaging contrast agent and an oral drug can be routinely determined by a clinician. The dosage regimen will depend on various factors such as whether the tumor or inflammatory lesion is diffuse or local, the health of the patient, sex and age, etc. By reference to other contrast agents and to the dosing regimen of the oral drug, one skilled in the art can determine the optimal effective dose and concentration of the drug of the present invention.

The drug-loaded microalgae can also be applied to intestinal radiation protection before radiotherapy of tumors of abdomen/basin; radiotherapy/chemotherapy of colon cancer is cooperated with tumor treatment; inflammatory bowel disease treatment, and the like.

The invention has the beneficial effects that the spirulina platensis is taken as the carrier of oral medicines, and has great potential of commercialization and clinical transformation. According to the invention, the small molecular medicine curcumin is loaded into the spirulina by using a one-step method, so that higher medicine loading efficiency is shown, the raw materials are easily available, the environment is protected, and the preparation method is simple.

In the aspect of oral treatment application, the curcumin-loaded spirulina platensis has a micron-sized dimension, the spiral form of the spirulina platensis is easy to be captured by intestinal villi, and long-time retention at intestinal tract parts can be realized. In addition, the microalgae carrier can obviously promote the absorption of curcumin and improve the bioavailability of the curcumin. Meanwhile, chlorophyll inherent in the spirulina carrier has natural fluorescence characteristics, can realize gastrointestinal tract fluorescence imaging after oral administration, and can realize noninvasive tracking in vivo without any extra fluorescent label. The drug delivery system has high drug carrying efficiency, good drug slow release property and biological safety, and can effectively realize the accurate treatment of colon cancer and colonitis under the guidance of fluorescence imaging.

Drawings

FIG. 1 is an optical microscope image and Scanning Electron Microscope (SEM) image of spirulina platensis Bright field (Bright-field) and fluorescent field (Fluorescence);

FIG. 2 is a graph showing statistics of curcumin-carrying efficiency of spirulina platensis at different material ratios;

FIG. 3 is a graph showing drug release at various time points of a curcumin-loaded spirulina platensis formulation after treatment with artificial gastric juice (SGF, a) and artificial intestinal juice (SIF, b);

FIG. 4 is a graph showing fluorescence signals of the gastrointestinal tract of mice at various time points before and after injection obtained by using a living body imager of a small animal after oral administration of a curcumin-loaded spirulina platensis preparation to the mice;

figure 5 morphological changes in the digestive tract after oral administration of curcumin-loaded spirulina platensis formulation in mice (bright field microscope, fluorescence field microscope and scanning electron microscope pictures).

FIG. 6 is a graph comparing the protective effect of curcumin-loaded spirulina platensis formulation, curcumin, spirulina platensis on impaired proliferation of the crypt of the small intestine due to X-ray abdominal irradiation (Ki 67 immunohistochemical staining).

Fig. 7 is a graph comparing tumor volume and weight results of a curcumin-loaded spirulina platensis formulation, curcumin, spirulina platensis after radiotherapy/chemotherapy co-treatment of cecum in situ colon cancer (p-value < 0.01; p-value < 0.001). In the figure, a is an in vitro photograph of cecum tissue with tumor, and b is a weight statistical image of tumor.

Fig. 8 is a graph comparing rectal bleeding after treatment of DSS-induced ulcerative colitis with curcumin-loaded spirulina platensis formulation, curcumin, spirulina platensis and colon length (5 replicates).

FIG. 9 is a graph comparing blood normals (WBC, white blood cells, RBC, red blood cells, HGB, hemoglobin, MCH, mean red blood cell hemoglobin, MCHC, mean red blood cell hemoglobin concentration, MCV, mean cell volume, PLT, platelets, HCT, hematocrit) and blood biochemical indicators (ALT, alanine transferase, AST, aspartate transferase, BUN, urea nitrogen, CREA, creatinine) after 30 days of continuous oral administration of curcumin-loaded spirulina platensis formulation.

Detailed Description

The present invention will be further described with reference to the following drawings and examples, but the present invention is not limited to the following examples.

Example 1 Synthesis of curcumin-carrying Spirulina platensis preparation SP@Curcumin

Taking 7 parts of Spirulina Platensis (SP) with 500 mug (dry weight) each, respectively suspending in 10mL of ultrapure water, correspondingly slowly adding 1mL of Curcumin (Curcumin) solution with the concentration of 25,50,100,200,400,800,1600 mug/mL respectively, and slowly stirring for 12 hours at room temperature and 180rpm in a dark place; finally, the mixture was centrifuged at 4500rpm for 10min, the supernatant was discarded, and the precipitate was suspended with ultrapure water to obtain curcumin-loaded spirulina platensis (SP@Curcumin). The drug loading efficiencies at different curcumin/spirulina material ratios were calculated by using the curcumin standard curve obtained by uv-vis absorption spectrum and the absorbance of different supernatants. As a result of calculating the drug loading efficiencies of different curcumin/spirulina material ratios, referring to fig. 2, it can be seen that when the curcumin/spirulina material ratio is greater than 0.4, the drug loading efficiencies of spirulina for loading curcumin are all above 80%.

Example 2 in vitro drug Release Performance test

The release profile of sp@curcumin (load factor 86.6%) in SGF containing Pepsin (Pepsin) at 37 ℃ and SIF containing pancreatin (Trypsin) at 37 ℃ was tested using uv-vis absorption spectroscopy; SP@Curcumin releases only a small amount of drug in a short time under SGF conditions, and releases only 8% of drug in 0.5 h. Under SIF conditions, drug release was up to 66% for 8h, as the result is seen in fig. 3.

EXAMPLE 3 fluorescence imaging Capacity and in vivo distribution

The whole body fluorescence imaging of mice after administration of SP@Curcumin intragastric drug was tested using a small animal living imager. 250 μL of SP@Curcumin (SP=1.7 mg/mL, curcumin=1.2 mg/mL) with a loading rate of 86.6% was injected into Balb/c nude mice by intragastric administration. After different times, signal patterns of the whole body of the mice are respectively obtained by using a living body imager of the small animals. As a result, referring to fig. 4, the fluorescence signal of the gastrointestinal tract is rapidly enhanced after injection of sp@curcumin compared to before injection, and the fluorescence signal is still detected at the intestinal tract after 24 hours, indicating that sp@curcumin has a better intestinal retention capacity, probably because its spiral form (fig. 1) is easily captured by intestinal villi, thereby achieving long-time retention at the intestinal tract. Meanwhile, the SP@Curcumin is proved to be used as an imaging agent for in-vivo fluorescence imaging.

EXAMPLE 4 in vivo degradation Properties

250 μL of SP@Curcumin (SP=1.7 mg/mL, curcumin=1.2 mg/mL) was injected into Balb/c nude mice by intragastric administration. After 2 hours, the contents of different parts of the gastrointestinal tract of the mice were taken out, and the morphology thereof was observed with an optical microscope and a scanning electron microscope. Results referring to fig. 5, sp@curcumin remained intact in the helical form in the stomach and duodenum, until the jejunum began to rupture, breaking into small pieces in the ileum and cecum. When reaching the colon, it breaks completely. The SP@Curcumin is gradually degraded after passing through the gastrointestinal tract environment, and the SP@Curcumin provides advantages for drug release in the intestinal tract and safety of in-vivo application of the SP@Curcumin.

EXAMPLE 5 intestinal radiation protection Properties

250 μL SP@Curcumin (SP=1.7 mg/mL, curcumin=1.2 mg/mL), 250 μL SP (SP=1.7 mg/mL), 250 μL Curcumin (Curcumin=1.2 mg/mL) were injected into Balb/c mice by intragastric administration. After 4h of administration, the mice were irradiated with X-rays at a dose of 12Gy, and after 7 days of irradiation, the ileal tissue of the mice was taken, and after 5% formaldehyde fixation, the mice were sectioned. Ileal sections were subjected to immunohistochemical (Ki 67) staining to evaluate the extent of radiation damage (proliferation crypt) of the small intestine. Results referring to fig. 6, sp@curcumin pretreated mice had the highest crypt proliferation activity. The SP@Curcumin is capable of reducing intestinal damage caused by high-dose radiation and effectively protecting intestinal tracts.

EXAMPLE 6 anti-colon cancer Properties

CT26-Luc colon cancer cells are inoculated on the cecal intestinal wall of Balb/c nude mice, and a cecal in-situ colon cancer animal model is constructed. After one week of molding, 250 μl of sp@curcumin (sp=1.7 mg/mL, curcumin=1.2 mg/mL), 250 μl of SP (sp=1.7 mg/mL), and 250 μl of Curcumin (curcumin=1.2 mg/mL) were injected into tumor-bearing Balb/c nude mice by intragastric administration, and after 4 hours of administration, the mice were subjected to X-ray irradiation at a dose of 12 Gy. And administered on days 3, 6, 9, 12 and 15, respectively, after irradiation. On day 15 post-irradiation, mice were euthanized and tumors were removed for measurement and weighing. Results referring to fig. 7, the sp@curcumin treated mice had significantly lower tumor volume and weight than the Control (cecal in situ colon cancer animal model) and other treated groups. The SP@Curcumin has good radiotherapy/chemotherapy synergistic effect, and can effectively inhibit the growth of colon tumor.

EXAMPLE 7 anti-colitis Properties

Balb/c mice were fed with 3% aqueous DSS for 10 days, a DSS-induced ulcerative colitis mouse model was constructed, and 250. Mu.L of SP@Curcumin (SP=1.7 mg/mL, curcumin=1.2 mg/mL), 250. Mu.L of SP (SP=1.7 mg/mL), and 250. Mu.L of Curcumin (Curcumin=1.2 mg/mL) were injected into the Balb/c mice on days 4, 7, 10, 13, and 16, respectively, by intragastric administration. On day 16, mice were assessed for rectal bleeding, euthanized, and the colon removed for measurement. Results referring to fig. 8, mice in the sp@curcumin treated group had slight rectal bleeding, and the colon length remained similar to that of the normal group, significantly superior to the other treated groups. The SP@Curcumin has good anti-inflammatory capability and can effectively relieve inflammatory reaction of colonitis.

EXAMPLE 8 oral safety

250 μL SP@Curcumin (SP=1.7 mg/mL, curcumin=1.2 mg/mL), 250 μL SP (SP=1.7 mg/mL), 250 μL Curcumin (Curcumin=1.2 mg/mL) were injected into Balb/c white mice by intragastric administration. After 30 days of administration, the blood of the mice is taken for routine blood detection and biochemical blood detection. Results referring to fig. 9, after administration of sp@curcumin, the major blood routine and blood biochemical index of the mice were both in the normal range, demonstrating that sp@curcumin has good oral safety.

Claims

1. A preparation method of a drug-loaded microalgae is characterized by comprising the following steps:

2. The method according to claim 1, wherein the ratio of the ultrapure water-suspended spirulina platensis algae solution to the curcumin solution is 50-500 μg/mL, 20-2000 μg/mL.

3. A drug-loaded microalgae prepared by the preparation method of any one of claims 1-2, the drug-loaded microalgae consisting of spirulina platensis and curcumin loaded in the spirulina platensis.

4. Use of a drug-loaded microalgae according to claim 3, comprising one or more of the following:

(1) Preparing contrast agent of stomach, ileum, cecum and colon tissues;

(2) Preparing oral medicine for colon cancer and/or colonitis;

(3) The intestinal radiation protective agent before radiotherapy of the tumor of the abdomen/basin is prepared.